The stability of the genome, which is dependent on faithful DNA replication, is essential to normal cell functions. Most human cancers have been shown to be related to some forms of genetic instabilities. Therefore, understanding the mechanism of DNA replication is fundamental in understanding the underlying causes of DNA instability related human diseases, as well as developing potential diagnostic and therapeutic approaches for these diseases. The objective of this research is to study the global organization of a replication fork, as the first step in understanding the structural basis for the coordinated synthesis of the leading and lagging strands during DNA replication. The bacteriophage T7 DNA replication system will be used as the model system in this study. Electron cryo-microscopic approaches will be employed to determine the structures of the complexes formed between replisomal proteins. The specific aims of this proposal are: 1. Determine the structures of the T7 gene 4 helicase/ primase and the T7 DNA polymerase complexes in the contexts of the leading and lagging strand synthesis, respectively, using electron cryomicroscopic (cryoEM) methods. 2. Determine the structure of the entire T7 phage replisome using cryoEM methods.